Bacillus velezensis and application of bacillus velezensis in rapidly increasing sauce flavor in salt-reduced soy sauce fermentation

Abstract

The invention discloses bacillus velezensis and application thereof in quickly increasing sauce flavor in salt-reduced soy sauce fermentation, and belongs to the technical field of food fermentation. The bacillus velezensis Bv-M1 is separated from soy sauce fermented mash for the first time, the bacillus velezensis strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32853. The TTMP content and the sauce flavor of the soy sauce added with the bacillus velezensis Bv-M1 aroma enhancement yeast extract liquid are obviously improved compared with the fermented soy sauce without addition, and the application example of the bacillus velezensis strain applied to soy sauce fermentation and sauce flavor enhancement is not reported yet at present.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food fermentation technology, and more particularly to a bacillus velezensis and its application in rapid increase of soy sauce flavor in salt-reduced soy sauce fermentation. BACKGROUND

[0002] The problem of insufficient soy sauce flavor at the end of traditional soy sauce fermentation process is often closely related to the lack of key flavor substances, i.e. pyrazine compounds. Pyrazine compounds (such as tetramethylpyrazine) are important contributors to the soy sauce flavor, and their content directly affects the aroma intensity and layering of soy sauce.

[0003] However, in the conventional brewing process, due to the metabolic limitations of the fermentation bacterial population or the inhibition of microbial activity in the high salt environment (usually 16-22% NaCl), the synthesis efficiency of pyrazine compounds is low, resulting in insufficient soy sauce characteristics of the finished product. In addition, the production of modern salt-reduced soy sauce (salt content ≤14%) further exacerbates this problem, as the reduction of salt content easily causes microbial spoilage during the fermentation of soy sauce, thereby degrading the microbial community structure and further limiting the accumulation of pyrazine compounds.

[0004] Currently, there are the following technical difficulties in the application of increasing soy sauce flavor in soy sauce brewing: first, there is no special endogenous flavoring bacteria of pyrazine soy sauce; second, there is a lack of process application for increasing the content of pyrazine compounds in soy sauce to increase soy sauce flavor.

[0005] Bacillus velezensis, as a gram-positive bacterium, has the ability to synthesize a variety of secondary metabolites. Studies have shown that it can synthesize flavor substances such as pyrazine, which provides the possibility for its directional strengthening of soy sauce flavor in salt-reduced fermentation of soy sauce.

[0006] In summary, how to provide a bacillus velezensis with high yield of pyrazine compounds and develop a process method for its application in salt-reduced soy sauce fermentation to increase soy sauce flavor is a problem that needs to be solved by those skilled in the art. SUMMARY

[0007] Therefore, the present application provides a bacillus velezensis and its application in rapid increase of soy sauce flavor in salt-reduced soy sauce fermentation. The flavoring koji extract of the bacillus velezensis Bv-M1 of the present application applied in soy sauce fermentation can significantly increase the content of tetramethylpyrazine (TTMP) in soy sauce, make the soy sauce rich in soy sauce flavor, and achieve rapid increase of soy sauce flavor and reduction of salt content.

[0008] To achieve the above purpose, the present application adopts the following technical solutions:

[0009] A strain of Bacillus velezensis, named Bv-M1, is classified as Bacillus velezensis, and is preserved in the China General Microbiological Culture Collection Center on November 29, 2024, with the preservation number CGMCC No. 32853 and the preservation address being No. 3, Beichen West Road, Chaoyang District, Beijing.

[0010] The above-mentioned Bacillus velezensis is applied in the fermentation production of TTMP.

[0011] The above-mentioned Bacillus velezensis is applied in the fermentation production of protease, amylase and cellulase.

[0012] The above-mentioned Bacillus velezensis is applied in the fermentation production of reduced-salt soy sauce.

[0013] The above-mentioned Bacillus velezensis is applied in the rapid increase of soy sauce aroma in soy sauce fermentation.

[0014] Further, the flavoring koji extract fermented and extracted by the above-mentioned Bacillus velezensis is added in the late stage of soy sauce fermentation.

[0015] Further, the preparation method of the flavoring koji extract is as follows:

[0016] (1) Prepare flavoring koji: mix steamed soybean meal and fried wheat in a mass ratio of 5-8:5-2, add water accounting for 10-50% of the total mass of raw materials, sterilize, cool to 40℃, inoculate the above-mentioned Bacillus velezensis, and inoculate 2-10%, ferment at 32-48℃ for 2-4 days;

[0017] (2) Extraction: add hot water at 50-70℃ to soak the finished flavoring koji prepared in step (1) for 1-2 hours according to the ratio of water to flavoring koji of 1-3:1 (w / w);

[0018] (3) Filtration: perform solid-liquid separation by filtration, and obtain the flavoring koji extract after membrane filtration of the liquid phase.

[0019] Further, the volume ratio of soy sauce mash to flavoring koji extract is 8-11:1.

[0020] Further, the flavoring koji extract is added to dilute the salt concentration of the soy sauce mash, so as to realize the reduced salt of the finished soy sauce.

[0021] The bacillus velezensis Bv-M1 of the present application metabolically accumulates acetoin, an important precursor of pyrazines, and further efficiently synthesizes pyrazines to enhance soy sauce flavor. In the soy sauce brewing process, whether inoculating the bacillus velezensis directly into the mash system for fermentation or adding the fermentation liquor of the bacillus velezensis, the flavor quality of the final product can be significantly improved. The present application adds the extraction liquor prepared by the flavor-enhancing koji of the bacillus velezensis filtered by a membrane at the end of the mash fermentation, which has multiple advantages: firstly, it can avoid the increase of sterilization difficulty caused by the introduction of bacillus; secondly, it can appropriately reduce the salt concentration of the finished soy sauce by adding the extraction liquor of the flavor-enhancing koji; and more importantly, it can significantly increase the content of pyrazines and enhance the typical soy sauce flavor. In addition, by adding the extraction liquor of the flavor-enhancing koji and continuing to ferment for a proper period of time, the flavor substances can be further integrated, and the single aroma component can be effectively avoided, so that the overall flavor is more coordinated and natural.

[0022] Through the above technical solutions, compared with the prior art, the present application has the following beneficial effects:

[0023] The bacillus velezensis Bv-M1 is isolated from the mash of soy sauce fermentation for the first time and used for producing the extraction liquor of flavor-enhancing koji. Compared with the fermentation soy sauce without adding the bacillus velezensis Bv-M1 extraction liquor of flavor-enhancing koji, the product has a significant increase in the content of TTMp and soy sauce flavor. At present, there is no reported example of the bacillus velezensis strain applied to the soy sauce fermentation for enhancing soy sauce flavor. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0025] Figure 1 The plate colony morphology (a) and the cell morphology (Gram staining, x1000) (b) of the strain Bv-M1 inoculated and cultured for 24h in Example 2 of the present application are shown in the following figure:

[0026] Figure 2 The electropherogram of the 16S rDNA sequence fragment in the molecular biology identification test of the strain Bv-M1 in Example 2 of the present application is shown in the following figure:

[0027] Figure 3 The phylogenetic tree in the molecular biology identification test of the strain Bv-M1 in Example 2 of the present application is shown in the following figure:

[0028] Figure 4This is a simplified process flow diagram of Bacillus vesiculosus Bv-M1 used in the fermentation of soy sauce in Example 5 of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0032] Example 1

[0033] Strain isolation and screening

[0034] 1. Initial screening of protease-producing strains

[0035] Weigh 30g of high-salt dilute fermented sauce mash samples with different fermentation times (2 months, 3 months, 4 months), add them to 100mL of sterile water under sterile conditions, mix well, treat in an 85℃ water bath for 10min, serially dilute and spread on 50g / L casein medium plates, incubate at 37℃ for 48h, and select strains with a large ratio of hydrolysis zone to colony diameter (D / d value) for further isolation and purification to obtain pure culture strains.

[0036] 2. Oxford cup method for secondary screening of strains with high protease, amylase, and cellulase activity.

[0037] Pure cultures of the bacterial strain were inoculated into 50 g / L beef extract peptone liquid medium and cultured at 37°C and 180 rpm for 24 h. The bacterial cells were then collected and prepared into 10... 8 A bacterial suspension of 1 cell / mL was prepared. 0.1 mL of the bacterial suspension was inoculated into an Oxford cup inserted in 50 g / L casein medium and incubated at 37°C for 48 h. The diameter of the hydrolysis zone was measured to screen for strains with high protease activity.

[0038] Selected high-protease-activity strains were inoculated into Oxford cups containing 50g / L salt-concentration starch medium according to the above method, and cultured at 37℃ for 48h. Iodine solution was sprayed onto the starch medium for color development, and the diameter of the starch hydrolysis zone was measured to screen strains with high amylase activity.

[0039] Selected high-amylase-activity strains were inoculated into Oxford cups placed on 50 g / L sodium carboxymethyl cellulose medium according to the above method, and cultured at 37°C for 48 h. 1 mg / mL Congo red solution was sprayed onto the sodium carboxymethyl cellulose medium and stained for 1 h. After washing with 1 mol / L NaCl solution for 1 h, the diameter of the cellulose hydrolysis zone was measured to screen strains with high cellulase activity.

[0040] After screening through the above process, a strain with high activity of protease, amylase, and cellulase was identified and named Bv-M1.

[0041] Example 2

[0042] Strain identification

[0043] 1. Morphological identification

[0044] (1) Strain Bv-M1 was inoculated onto high-salt beef extract peptone agar plates and cultured at 32°C for 24 hours. After the colony surface dried, wrinkled protrusions appeared. Figure 1 a).

[0045] The preparation steps for high-salt beef extract peptone culture medium are as follows: prepare 5g beef extract, 10g peptone, 180g sodium chloride, 15-20g agar, 1000mL distilled water, pH 7.0; sterilize at 121℃ for 30min.

[0046] (2) Gram staining of bacterial colonies of strain Bv-M1 and observation of the morphological characteristics of the strain under an oil microscope revealed that this bacterium is Gram-positive, has spores, and the individual bacterial cells are rod-shaped, with a size of approximately 0.5 × 2 μm to 0.5 × 3 μm. Figure 1 b).

[0047] 2. Molecular biological identification

[0048] (1) Primer design:

[0049] PCR amplification of 16S rDNA was performed using universal bacterial primers. The primer design is as follows:

[0050] Forwardprimer: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 1;

[0051] Reverse primer: 5'-AAGGAGGTGATCCAGCCGCA-3', SEQ ID No. 2.

[0052] The distance between the two primers is approximately 1500 bp.

[0053] (2) DNA template preparation:

[0054] A small amount of bacterial cells were picked from the cultured strain Bv-M1 slant and placed in a 50 μL colony PCR template extraction buffer system. The cells were denatured at 80 °C for 15 min and centrifuged at 4000 r / min for 10–15 min. The supernatant was then used as the DNA template.

[0055] (3) PCR to obtain 16S rDNA fragment:

[0056] Reaction system: The 50 μL reaction system contains 25 μL of PCR Premix, 0.5 μL of Forward Primer (20 pmol / μL), 0.5 μL of Reverse Primer (20 pmol / μL), 1 μL of template, and 23 μL of 16S-free H2O.

[0057] Reaction conditions: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 1.5 min, 30 cycles; final extension at 72℃ for 5 min.

[0058] (4) Electrophoresis:

[0059] The PCR product results were detected by agarose gel electrophoresis.

[0060] Electrophoresis conditions: 90V constant voltage, time 20-30 min, results are shown below. Figure 2 .

[0061] (5) 16S rDNA sequence sequencing

[0062] The 16S rDNA gene sequence of strain Bv-M1 is shown in SEQ ID No. 3.

[0063]

[0064] (6) Comparison of sequencing results

[0065] Sequencing results were analyzed for sequence homology using the Blast search database in GenBank, and a phylogenetic tree was constructed using MAGE 10.0 software as follows: Figure 3 As shown.

[0066] The results showed that strain Bv-M1 was most closely related to Bacillus velezensis, with a similarity of 100%.

[0067] Example 3

[0068] Preservation of Bacillus belye Bv-M1

[0069] Bacillus velezensis Bv-M1, classified as Bacillus velezensis, was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32853, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0070] Example 4

[0071] Fermentation characteristics of Bacillus belye Bv-M1

[0072] Bacillus belye Bv-M1 has the ability to secrete highly active proteases, amylases and cellulases, and can also efficiently synthesize acetoin and tetramethylpyrazine, precursors of tetramethylpyrazine (TTMP).

[0073] 1. Preparation of the test solution

[0074] The selected Bacillus strains were inoculated into beef extract peptone medium and cultured at 37°C and 180 rpm in a shaker to prepare a 10% concentration. 8 A bacterial suspension of 1 cell / mL was inoculated into the koji-making medium at an inoculum size of 2% (v / w). The koji was made at 37°C for 84 h. Samples taken at 0, 12, 24, 36, 48, 60, 72, and 84 h were extracted with hot water at 60°C to obtain the test solutions. The activities of protease, amylase, and cellulase, as well as the accumulation of acetoin and tetramethylpyrazine, were determined.

[0075] 2. Protease activity

[0076] (1) Determination of standard curve: Prepare a concentration gradient of standard tyrosine solution (50 μg / mL), add 0.55 mol / L sodium carbonate and Folin-phenol reagent, measure absorbance at 680 nm, and plot the standard curve.

[0077] (2) Enzyme reaction: The test solution was reacted with 0.5% casein solution at 30℃ for 10 min, and 10% trichloroacetic acid was added to terminate the reaction. The precipitate was removed by filtration.

[0078] (3) Colorimetric determination: Add sodium carbonate and Folin-phenol reagent to the filtrate, and measure the absorbance at 680 nm after color development. Calculate the amount of tyrosine produced based on the standard curve.

[0079] (4) Enzyme activity calculation: The enzyme activity unit (U / g) is defined as the amount of enzyme that catalyzes the production of 1 μg of tyrosine per minute, and the result is calculated on a dry basis.

[0080] As shown in Table 1, the protease activity of Bacillus belye Bv-M1 was the highest after 60 hours of fermentation, at 2469.47 U / g.

[0081] 3. Amylase activity

[0082] (1) Determination of standard curve: Prepare a concentration gradient of standard glucose solution (2 mg / mL), add DNS reagent, boil in water for 5 min for color development, and measure absorbance at 540 nm after cooling to plot the standard curve.

[0083] (2) Enzyme reaction: The test solution was reacted with 1% soluble starch solution at 37℃ for 5 min, DNS reagent was added to terminate the reaction and color development was observed, and the solution was cooled after boiling in a water bath for 5 min.

[0084] (3) Absorbance determination: The absorbance of the reaction solution was measured at 540 nm, and the content of reducing sugar (calculated as glucose) generated was calculated according to the standard curve.

[0085] (4) Enzyme activity calculation: The unit of amylase activity (U / g) is defined as the amount of enzyme required to catalyze the production of 1 mg of reducing sugar per minute, and the result is calculated on a dry basis.

[0086] As shown in Table 1, Bacillus belye Bv-M1 exhibited the highest amylase activity, reaching 74.18 U / g, after 72 hours of fermentation.

[0087] 4. Cellulase activity

[0088] (1) Preparation of standard curve: Prepare a concentration gradient of standard glucose solution (2 mg / mL), add DNS reagent, boil in water for 5 min for color development, and measure absorbance at 540 nm after cooling to plot the standard curve.

[0089] (2) Enzyme reaction: The test solution was reacted with 1% sodium carboxymethyl cellulose in a water bath at 50°C for 30 min. DNS reagent was added to terminate the reaction and develop color. The solution was then cooled after boiling in a water bath for 5 min.

[0090] (3) Absorbance determination: The absorbance of the reaction solution was measured at 540 nm, and the content of reducing sugar (calculated as glucose) generated was calculated according to the standard curve.

[0091] (4) Enzyme activity calculation: The unit of amylase activity (U / g) is defined as the amount of enzyme required to catalyze the production of 1 mg of reducing sugar per minute, and the result is calculated on a dry basis.

[0092] As shown in Table 1, Bacillus belye Bv-M1 exhibited the highest cellulase activity, reaching 9.25 U / g, after 60 hours of fermentation.

[0093] 5. Accumulated amount of marriage for spouse B

[0094] (1) Determination of standard curve: Prepare a concentration gradient of acetoin glucose standard solution (50.00 mg / L), add creatine colorimetric reagent, react in a 40℃ water bath for 30 min, measure absorbance at 510 nm wavelength, and plot the standard curve.

[0095] (2) Sample preparation: Mix the test solution with creatine colorimetric reagent and develop the color at 40℃ for 30 min.

[0096] (3) Absorbance measurement and acetoin concentration calculation: The absorbance at 510 nm was measured immediately after the reaction was completed. The concentration of acetoin in the sample was calculated according to the standard curve and converted on a dry basis.

[0097] As shown in Table 1, the accumulation of acetoin in the Bacillus belye Bv-M1 fermentation product reached a maximum of 42.06 g / kg after 36 h of fermentation.

[0098] 6. Tetramethylpyrazine accumulation

[0099] (1) Standard curve determination: A concentration gradient of TTMP standard solution (1.00 g / L) was prepared, and 5 μL of 0.816 μg / μL 2-methyl-3-heptanone was added as an internal standard. The results were measured on an SPME-GC-MS instrument. SPME conditions: shaker 50℃, sample heated and shaken for 5 min, adsorption extraction for 20 min, and elution for 5 min; GC conditions: injection port temperature 250℃, programmed temperature rise, 40℃ held for 4 min, temperature increased at 5℃ / min to 120℃, held for 2 min, then increased at 15℃ / min to 230℃, held for 4 min, carrier gas was high-purity helium, flow rate was 1.2 mL / min, split ratio was 10:1; MS conditions: EI ion source, electron energy 70 eV, emission current 200 μA, ion source temperature 250℃, mass scan range m / z 30-500.

[0100] (2) Sample preparation: Take 2 mL of the test solution and add it to a 20 mL headspace vial. Adjust the NaCl concentration to 30% and add 5 μL of 0.816 μg / μL 2-methyl-3-heptanone as an internal standard. Measure the sample on an SPME-GC-MS instrument.

[0101] (3) TTMP concentration calculation: The mass concentration of TTMP in the sample is calculated by using the standard curve, and the results are on a dry basis.

[0102] As shown in Table 1, the accumulation of TTMP in the Bacillus belyss Bv-M1 fermentation product reached a maximum of 608.73 mg / kg after 72 h of fermentation.

[0103] Table 1. Determination of protease activity, amylase activity, cellulase activity, acetoin, and TTMP accumulation.

[0104]

[0105]

[0106] Example 5

[0107] Bacillus vesiculosus Bv-M1 is used in the fermentation of soy sauce.

[0108] A simplified process flow diagram of using Bacillus vesiculus Bv-M1 in the fermentation of soy sauce is shown below. Figure 4 As shown. The specific process operation is as follows:

[0109] 1. Bacillus belye Bv-M1 flavoring extract

[0110] (1) Slant culture: Prepare beef extract peptone solid medium, inoculate and culture Bacillus belye Bv-M1, culture time is 15h, culture temperature is 36℃.

[0111] (2) Erlenmeyer flask culture: Bacillus vegetans Bv-M1 with good growth on the slant was inoculated into beef extract peptone liquid medium and cultured for 15 hours at a temperature of 36°C.

[0112] (3) Seed tank culture: After cleaning the seed tank, add beef extract peptone liquid culture medium and sterilize it (the volume of liquid culture medium is 60% of the volume of the seed tank). After cooling, inoculate with Bacillus belye Bv-M1, expand the culture time to 15h, the culture temperature is 36℃, and the stirring speed is 160rpm to obtain the seed liquid.

[0113] The sterilization conditions for the above-mentioned slant culture, Erlenmeyer flask culture and seed tank culture were all 121℃ for 30 min.

[0114] (4) Preparation of aroma-enhancing koji: Mix cooked soybean meal and roasted wheat in a certain ratio of 7:3 (w / w), and transport them to a disc koji-making machine through a screw conveyor. Add water of 20% of the total mass of raw materials, sterilize, cool to 40°C, inoculate with seed liquid of Bacillus belye Bv-M1 at a ratio of 10% (v / w), and ferment at 36°C for 3 days.

[0115] (5) Extraction: Add 60℃ hot water to the flavoring koji prepared in step (4) for 1 hour at a ratio of 2:1 (w / w) to extract the characteristic substances of soy sauce flavor - pyrazine compounds.

[0116] (6) Filtration: The solid and liquid phases are separated by plate and frame filter press. The solid phase is returned to the fermentation system for recycling, and the liquid phase is filtered through a membrane to obtain the flavor-enhancing extract.

[0117] 2. High-salt, low-temperature fermentation of soy sauce

[0118] (1) Mix cooked soybean meal and roasted wheat in a certain ratio of 7:3 (w / w), inoculate with Aspergillus oryzae 3.042 at a ratio of 0.2% (w / w), and ventilate at 32℃ for 36 hours to obtain the finished koji.

[0119] (2) Mix the finished koji with 23% brine at a ratio of 1:2 (w / v) and carry out the main fermentation at 28°C for 5 months.

[0120] 3. Preparation of Salt-Reduced and Flavor-Enhanced Soy Sauce

[0121] (1) During the later stage of high-salt dilute fermentation of soy sauce (140 days), add flavoring yeast extract at a ratio of 8:1 between the volume of soy sauce mash and flavoring yeast extract, and continue fermentation for 10 days.

[0122] (2) After adding the flavor-enhancing yeast extract, the fermented mash that has been fermented for 150 days is pressed to obtain a flavor-coordinated low-sodium flavor-enhancing soy sauce.

[0123] Example 6

[0124] Optimization of the process for producing flavor-enhancing koji using Bacillus belysae Bv-M1

[0125] Based on the characteristics of Bacillus belyssus Bv-M1, the koji-making process of Bacillus belyssus Bv-M1 was systematically optimized to increase the accumulation of tetramethylpyrazine (TTMP) in the aroma-enhancing koji extract. The specific experimental design and results are as follows:

[0126] The process for preparing the aroma-enhancing yeast is as described in Example 5.

[0127] The extraction and filtration steps for preparing the flavor-enhancing yeast extract are the same as in Example 5.

[0128] The method for determining tetramethylpyrazine (TTMP) is the same as in Example 4.

[0129] (1) Optimization of roasted wheat addition: Based on the quality of soybean meal, the roasted wheat addition was set to 40%, 50%, 60%, 70%, and 80% (w / w), with a fixed water addition of 20%, inoculum amount of 2% (v / w), temperature of 37℃, fermentation for 3 days, and the experiment was repeated three times.

[0130] The results in Table 2 show that when the amount of roasted wheat added was 50%, the TTMP yield reached a peak of 617.68 mg / kg. Excessive roasted wheat (>60%) inhibited the metabolic activity of the microorganisms and reduced the TTMP yield.

[0131] Table 2 Results of Optimization of Roasted Wheat Addition Amount

[0132] Fried wheat addition amount 80％ 70％ 60％ 50％ 40％ TTMP (mg / kg dry basis) 361.56±5.77 432.72±2.06 561.72±51.90 617.68±29.13 432.79±9.57

[0133] (2) Optimization of water addition: The amount of roasted wheat added was fixed at 50% (w / w), and the amount of water added was adjusted to 10%, 20%, 30%, 40% and 50% (v / w) of the total mass of soybean meal-roasted wheat. The inoculum amount was 2% (v / w), the temperature was 37℃, and the fermentation was carried out for 3 days. The experiment was repeated three times.

[0134] The results in Table 3 show that the highest accumulation of TTMP (650.97 mg / kg) was observed when the water content was 20%. Too low a water content (10%) resulted in limited cell growth, while too high a water content (>30%) diluted the substrate concentration.

[0135] Table 3 Results of Water Addition Optimization

[0136] Water addition amount 10％ 20％ 30％ 40％ 50％ TTMP (mg / kg dry basis) 386.60±11.59 650.96±9.50 595.78±17.85 491.53±10.15 422.46±5.02

[0137] (3) Optimization of inoculum amount: Under the conditions of 50% roasted wheat addition, 20% water addition, and 37℃ temperature, the effect of inoculum amount (2%, 4%, 6%, 8%, 10%, v / w) was investigated. Fermentation lasted for 3 days, and the experiment was repeated three times.

[0138] The results in Table 4 show that TTMP production reached its highest level (1273.82 mg / kg) when the inoculum size was 8%. However, when the inoculum size exceeded 8%, bacterial competition intensified, metabolic byproducts increased, and TTMP production decreased.

[0139] Table 4 Results of Optimized Vaccination Volume

[0140] Inoculum amount 2％ 4％ 6％ 8％ 10％ TTMP (mg / kg dry basis) 610.42±3.16 905.02±47.09 1070.12±37.34 1273.82±36.63 1122.00±30.20

[0141] (4) Fermentation temperature optimization: With fixed addition of 50% roasted wheat, 20% water, and 8% inoculum, temperature gradients (32, 36, 40, 44, 48℃) were tested, fermentation was carried out for 3 days, and the experiment was repeated three times.

[0142] The results in Table 5 show that the highest accumulation of TTMP (1817.50 mg / kg) was observed at 40℃.

[0143] Table 5 Results of Fermentation Temperature Optimization

[0144] Culture temperature 32℃ 36℃ 40℃ 44℃ 48℃ TTMP (mg / kg dry basis) 901.95±62.33 1336.33±68.56 1817.5±37.97 1465.21±30.92 1361.81±14.92

[0145] Example 7

[0146] Fermentation process optimization and effect verification

[0147] The aroma-enhancing koji extract obtained based on the optimal parameters of Example 6 was used to verify its effect on increasing the TTMP content of soy sauce after fermentation. The specific implementation process is as follows:

[0148] In the later stage of high-salt dilute soy sauce fermentation, flavor enhancer extract was added to the fermentation system at a ratio of 8:1 (v / v) of soy sauce mash to flavor enhancer extract. Fermentation continued at 28℃ for 10 days. The difference in TTMP content between the added group and the normally fermented soy sauce (without addition) was analyzed and compared.

[0149] Table 6 shows that the TTMP content of soy sauce with added Bacillus vesiculosus Bv-M1 flavor enhancer extract and normally fermented soy sauce were analyzed. The TTMP content of soy sauce with added flavor enhancer extract reached 106.78 mg / L, which was significantly higher than 12.22 mg / L of normally fermented soy sauce, with an increase of 773.83%. At the same time, the salt content was reduced to 14.22%, while the amino acid nitrogen content remained within the range of premium soy sauce (≥8 g / L), achieving the dual effect of reducing salt and enhancing soy flavor.

[0150] Table 6 Physicochemical Properties of Soy Sauce

[0151]

[0152] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0153] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus belesii, characterized in that, Named Bv-M1, and classified as Bacillus velezensis, it was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32853, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of Bacillus belye as described in claim 1 in the fermentation production of tetramethylpyrazine.

3. The application of Bacillus belye as described in claim 1 in the fermentation production of protease, amylase, and cellulase.

4. The application of Bacillus berberis as described in claim 1 in the fermentation production of reduced-sodium soy sauce.

5. The application of Bacillus vesiculosus of claim 1 in rapidly enhancing the flavor of soy sauce during salt reduction fermentation.

6. The application as described in claim 5, characterized in that, Add the flavor-enhancing extract obtained by fermentation and extraction of Bacillus vesiculosus as described in claim 1 during the later stage of soy sauce fermentation.

7. The application as described in claim 6, characterized in that, The preparation method of the flavor-enhancing yeast extract is as follows: (1) Preparation of aroma-enhancing starter: Mix cooked soybean meal and roasted wheat in a mass ratio of (5-8):(5-2), add water of 10-50% of the total mass of raw materials, sterilize and cool to 40°C, inoculate with the Bacillus belye at an inoculation amount of 2-10%, and ferment at 32-48°C for 2-4 days; (2) Extraction: Add hot water at 50-70℃ and soak the finished flavoring koji prepared in step (1) for 1-2 hours at a mass ratio of water to flavoring koji of 1-3:

1. (3) Filtration: Filtration is used to separate solid and liquid phases. The liquid phase is filtered through a membrane to obtain the flavoring extract.

8. The application as described in claim 6, characterized in that, The volume ratio of the fermented sauce to the flavoring yeast extract is 8–11:

1.

9. The application as described in claim 8, characterized in that, Adding flavor-enhancing yeast extract is used to dilute the salt concentration of the fermented soybean mash, thereby reducing the salt content of the finished soy sauce.

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